Adipocytes are highly specialized cells that play a critical role in energy and homeostasis. Their primary role is to store triglycerides in times of caloric excess and to mobilize this reserver during periods of nutritional deprivation. Adipocytes are derived from a multipotent stem cell of mesodermal origin that also gives rise to the muscle and cartilage lineages. Adipocyte differentiation is characterized by a coordinate increase in adipocyte-specific gene expression.
Recent years have seen important advances in our understanding of the molecular basis of adipocyte differentiation. (reviewed in Cornelius, P. et al. (1994) Annu. Rev. Nutr. 14:99-129; Tontonoz, P. et al. (1995) Curr. Opin. Genet. Dev. 5:571-576. A number of transcription factors are induced in fat cell differentiation (C/EBPxcex1, C/EBPxcex2 and ADD1/SREBP1) and influence this process to a certain extent (Freytag, S. O. et al. (1994) Genes Dev. 8:1654-63; Kim, J. B. and Spiegelman, B. M. (1996) Genes Dev. 10:1096-1107; Lin, F. T. and Lane, M. D. (1994) PNAS USA 91:8757-61; Samuelsson, L. et al. (1991) EMBO J. 10:3787-93; Tontonoz, P. et al. (1993) Mol Cell Biol 13:4753-9; Umek, R. M. et al. (1991) Science 251:288-92; Wu, C. L. et al. (1995) Mol Cell Biol 15:253646; Yeh, W. C. et al. (1995) Genes Dev. 9:168-81).
The peroxisome proliferator-activated receptors, or xe2x80x9cPPARxe2x80x9d, are members of the type II class of steroid/thyroid superfamily of receptors and which mediate the pleiotropic effects of peroxisome proliferators. Type II class of nuclear receptors includes PPAR, the thyroid hormone receptor (T3R), and the vitamin D3 receptor (VD3R). Type II receptors are functionally distinct from the classical steroid receptors, such as the glucocorticoid receptor, the progesterone receptor and the estrogen receptor (reviewed in Stunnenberg, H. G. (1993) Bio Essays Vol. 15 (5): 309-15. Three properties distinguish these two classes. Firstly, type II receptors are able to bind to their responsive elements in the absence of ligand (Damm et al. (1989) Nature 339:593-597; Sap et al., Nature 340:242-244; De The et al. (1990) Nature 343:177-180), whereas ligand binding is required to dissociate to the type I receptor-hsp 90 complex and hence indirectly governs DNA binding. Secondly, type II receptors bind and transactivate through responsive elements that are composed of half-sites arranged as direct repeats, as opposed to palindromically arranged half-sites invariably separated by three nucleotides required by type I receptors. Finally, type II receptors do not bind to their respective binding site as homodimers but require an auxiliary factor, RXR (e.g., RXRxcex1, RXRxcex2, RXRxcex3) for high affinity binding (Yu et al. (1991) Cell 67:1251-1266; Bugge et al. (1992) EMBO J. 11:1409-1418; Kliewer et al. (1992) Nature 355:446-449; Leid et al. (1992) Cell 68:377-395; Marks et al. (1992) EMBO J. 11:1419-1435; Zhang et al. (1992) Nature 355:441-446). The interaction between type II receptors requires a region in the C-terminal domain (Yu et al. (1991) Cell 67:1251-1266; Kliewer et al. (1992) Nature 355:446-449; Leid et al. (1992) Cell 68:377-395; Marks et al. (1992) EMBO J. 11:1419-1435). Following binding, the transcriptional activity of a target gene (i.e., a gene associated with the specific DNA sequence) is enhanced as a function of the ligand bound to the receptor heterodimer.
The present invention is based on the finding that activation of PPARxcex3 plays a key role in inducing growth arrest by terminal differentiation of actively proliferating PPARxcex3-expressing cells, particularly transformed adipose precursor cells.
Accordly, one aspect of the invention provides a method for inhibiting proliferation of a PPARxcex3-responsive hyperproliferative cell, comprising ectopically contacting the cell with a a PPARxcex3 agonist in an amount effective to induce differentiation of the cell. For example, the instant method can be used for the treatment of, or prophylactically prevention of a disorder characterized by aberrant cell growth of PPARxcex3-responsive hyperproliferative cells, e.g., by administering a pharmaceutical preparation of a PPARxcex3 agonist in an amount effective to inhibit growth of the PPARxcex3-responsive hyperproliferative cells.
For example, the subject method can be used in the treatment of sarcomas, carcinomas and/or leukemias. Exemplary disorders for which the subject method may be used as part of a treatment regimen include: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing""s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms"" tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.
In certain embodiments, the subject method can be used to treat such disorders as carcinomas forming from tissue of the breast, prostate, kidney, bladder or colon.
In other embodiments, the subject method can be used to treat hyperplastic or neoplastic disorders arising in adipose tissue, such as adipose cell tumors, e.g., lipomas, fibrolipomas, lipoblastomas, lipomatosis, hibernomas, hemangiomas and/or liposarcomas.
In still other embodiments, the subject method can be used to treat hyperplastic or neoplastic disorders of the hematopoietic system, e.g., leukemic cancers. In a preferred embodiment, the subject is a mammal, e.g., a primate, e.g., a human.
In preferred embodiments, the PPARxcex3 agonist used in the instant method is a ligand of a PPARxcex3 protein which activates a transcriptional activity of the PPARxcex3 protein. For example, the PPARxcex3 agonist can be a thiazolidinedione, or an analog thereof. Exemplary PPARxcex3 agonists include pioglitazone, troglitazone, ciglitazone, englitazone, BRL49653, and chemical derivatives thereof. In certain preferred embodiments, the PPARxcex3 agonist is represented in the general formula: 
or a tautomeric form thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, in which A1 represents a substituted or unsubstituted aromatic heterocyclyl group; R1 represents a hydrogen atom, an alkyl group, an acyl group, an aralkyl group, wherein the aryl moiety may be substituted or unsubstituted, or a substituted or unsubstituted aryl group; R2 and R3 each represent hydrogen, or R2 and R3 together represent a bond; A2 represents a benzyl or chromanyl moiety having, as valence permits, up to five substituents; and n represents an integer in the range of from 1 to 6.
In other embodiments, the PPARxcex3 agonist can be a naturally-occurring ligand of the receptor, such as an arachidonate metabolite, e.g., a metabolite of PGD2.
In order to avoid or minimize certain unwanted side-effects to treatment with a PPARY agonists, it may be desirable in certain embodiments of the subject method that the PPARxcex3 agonist activates PPARxcex3-dependent transcription at a concentration at least one order of magnitude less than required for the same level of activation of PPARxcex1, PPARxcex4 or RaR-dependent transcription.
The PPARxcex3 agonist can be administered alone, or as part of a combinatorial therapy. For example, the PPARxcex3 agonist can be conjointly administered with one or more agents such as mitotic inhibitors, alkylating agents, antimetabolites, nucleic acid intercalating agents, topoisomerase inhibitors, agents which promote apoptosis, and/or agents which increase immune responses. In other embodiments, the PPARxcex3 agonist can be conjointly administered with an RxR agonist. Such RxR agonist can be natural or synthetic retinoids. An exemplary RxR agonist is represented in the general formula: 
Still another aspect of the present invention provides compositions and kits for conjointly administering a PPARxcex3 agonist and an RxR agonist. For example, both agents can be pre-mixed, preferably in a pharmaceutically acceptable carrier. Alternatively, the agents can be provided separately in the form of a kit comprising (i) a first pharmaceutical composition including a PPARxcex3 ligand in a pharmaceutically acceptable carrier, and (ii) a second pharmaceutical composition including an RxR agonists in a pharmaceutically acceptable carrier, the PPARxcex3 and RxR agonists being present in a therapeutically effective amount to, upon conjoint administration, induce terminal differentiation of a PPARxcex3-responsive hyperproliferative cell in a subject animal.
Likewise, the PPARxcex3 agonist useful in the methods of the present invention can be administered conjointly with other agents which effect, e.g., the growth of, or immune response against, the hyperproliferative cells to be treated. As above, the secondary agents can be pre-mixed with the PPARxcex3 agonist, or provided as part of a kit comprising (i) a first pharmaceutical composition including a PPARxcex3 ligand in a pharmaceutically acceptable carrier, and (ii) a one or more additional pharmaceutical composition(s) including one or more agents selected from the group consisting of mitotic inhibitors, alkylating agents, antimetabolites, nucleic acid intercalating agents, topoisomerase inhibitors, agents which promote apoptosis, and agents which increase immune responses to tumors.
This invention also relates to the surprising discovery that PPARxcex3 is consistently and selectively expressed in each of the major histologic types of human liposarcoma compared to other soft tissue sarcomas. Accordingly, another aspect of the present invention provides a method for augmenting diagnosis of liposarcomas, comprising detecting in a sample of transformed cells one or both of a diagnostic level of PPARxcex3 mRNA or PPARxcex3 protein, wherein elevated expression of PPARxcex3 mRNA or protein in cells of the sample increases the likelihood that at least a portion of the transformed cells of the sample are liposarcoma cells. For example, the diagnostic assay can be carried out on a biopsy obtained from a soft tissue hyperplasia or neoplasia.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning, A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.